Molecular Mechanisms Of Sustained Autophagy Regulation By Peste Des Petits Ruminants Virus In Host Cells

Peste des petits ruminants virus?PPRV?,the etiological agent of peste des petits ruminants?PPR?,causes an acute or subacute disease in small ruminants.Its outbreak was first reported in 2007 and causes serious economic losses in China.Currently,vaccination is the most effective method to control the disease.PPRV has a well-established lymphatic and epithelial tissue tropism,which mainly affects lymphatic tissue,respiratory system and digestive system.Although abortion is observed in an unusually large proportion of pregnant goats during outbreaks of PPR,the pathogenic mechanism underlying remains unclear.Autophagy is a lysosomal dependent degradation pathway which plays an important role in the maintenance of intracellular homeostasis.Autophagy is a double-edged sword.It delivers misfolded or long-lived cytoplasmic proteins and damaged organelles to lysosomes for degradation and recycling.An increasing amount of evidences suggest that the autophagy processes are also exploited by many viruses for their replication.However,the relationship between PPRV and autophagy in host cells has not been reported.In this study,we investigated the changes of intracellular molecules and the relationship between PPRV and cellular autophagy,the research contents and results of this project are as follow:?1?Screening and analysis of differentially expressed genes during PPRV infection in host cells.Here,the gene expression profile of caprine endometrial epithelial cells?EECs?infected with PPRV Nigeria 75/1 was determined by DNA microarray to investigate the gene expression pattern and the cellular response immediately after viral entry.The microarray analysis revealed that a total of 146 genes were significantly dysregulated by PPRV internalization within 1 h post-infection?hpi?.Of these,85 genes were upregulated and 61genes were downregulated.Most of these genes,including NF?B1A,JUNB,and IL-1A,have not previously been reported in association with PPRV infection in goats.Following viral replication?24 hpi?,the expression of 307 genes were significantly upregulated and that of 261 genes were downregulated.The data for the genes differentially expressed in EECs were subjected to a time sequence profile analysis,gene network analysis and pathway analysis.Pathway analysis revealed that these differentially expressed genes were mainly involved in anti-inflammatory response,cell cycle,mTOR signaling Pathway,apoptosis signaling Pathway,MAPK signaling Pathway,etc.The gene network analysis showed that13 genes?EIF2AK3,IL-10,TLR4,ZO3,NF?BIB,RAC1,HSP90AA1,SMAD7,ARG2,JUNB,ZFP36,APP,and IL-1A?were located in the core of the network.The differentially expressed genes such as TNF,NF?BIB,IL-1 and TLR4 were verified by real-time PCR and the accuracy of DNA microarray was determined.In addition,studies have shown that PPRV infected EECs significantly up-regulates the expression of Nectin-4 and plays a key role on its cellular localization.In this study,we screened out the differentially expressed genes when PPRV binds EECs,providing theoretical and scientific basis for virus invasion and early infection.?2?The effect of PPRV induced EECs autophagy on virus replication.In this study,the results of transmission electron microscopy and western blot confirmed that autophagy was induced upon virus infection in EECs,as determined by the appearance of double-and single-membrane autophagy-like vesicles and LC3-I/LC3-II conversion,indicating that PPRV can induce autophagy in host cells.In addition,a significant decrease in p62 protein levels was detected in PPRV-infected cells.E64d causes the increase of p62 and the accumulation of LC3-II during PPRV infection,indicating that PPRV can trigger a complete autophagy response.To further analyse the effect of various viral proteins on autophagy induction during PPRV infection,we expressed N,F,C,H and V as fusion proteins with the HA protein,respectively.Interestingly,HA-N,HA-C and HA-H increased the levels of LC3-II and were highly co-localized with the punctate LC3-positive fluorescent staining,indicating that PPRV non-structural and structural protein induces autophagy in EECs.To further analyse the role of autophagy in PPRV replication,we examined viral protein N expression and the viral progeny yields following Rapamycin,NH₄Cl,Chloroquine,Wortmannin,and specific shRNAs targeting Beclin-1 and ATG7 treatment.Importantly,the inhibition of autophagosome formation with Wortmannin,specific shRNAs targeting Beclin-1 and ATG7 induced caspase-dependent apoptosis in PPRV-infected EECs.However,the inhibition of autophagy with NH₄Cl or Chloroquine did not increase the number of apoptotic cells or the activity of caspase.Collectively,these data are the first to indicate that PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication in host cells.?3?The molecular mechanism of Nectin-4 mediates autophagy and PPRV-induced sustained autophagy.In this study,EECs were infected with PPRV for 0,1.5,3,6,9,12 and24 h.We confirmed that PPRV infection induces two successive autophagic signalling in host cells via distinct and uncoupled molecular pathways.Immediately upon invasion,PPRV induces a first transient wave of autophagy via a pathway involving its cellular receptor Nectin-4 and the AKT-mTOR dependent pathway.Autophagic detection showed that early PPRV infection not only increased the amount of autophagosomes and light chain 3?LC3?-II but also downregulated AKT-mTOR phosphorylation.Further studies have shown that the viral protein H binds to Nectin-4 is a necessary condition to induce the first wave of autophagy,indicating that autophagy is a key pathway for cells to resist virus infection.The first wave of autophagy was maintained for a short time and recovered to the basic level after PPRV infection for 3 h.Soon after infection,new autophagic signalling was initiated that required viral replication and protein expression.Moreover,GFP-LC3 EECs were treated with si-IRGM or si-HSP70 significantly reduced the expression of LC3-II and the number of GFP⁺-LC3 vesicles per cell profile,which revealed that IRGM-interacting PPRV-C and HSP70-interacting PPRV-N expression is sufficient to induce autophagy through an IRGM-HSP70 dependent pathway.In conclusion,we used DNA microarray technology to screen out the intracellular genes during PPRV infection in host EECs,providing a theoretical basis for the interaction between PPRV and host cells.Importantly,PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication and maturity in host cells.Overall,our work reveals distinct molecular pathways for the induction of self-beneficial sustained autophagy by attenuated PPRV,which contributes to controlling viral infection and better usage of vaccines for therapy.